Mass spectrometry techniques have been used to produce sequence information by fragmenting peptide ions. One conventional method is collision-induced dissociation. However, with this method, multiple fragment types are often produced (mostly b and y type), the sequence coverage may vary widely, and the observed fragmentation often cannot be readily predicted from the peptide sequence. For example, Scheme 1 shows (a) the standard nomenclature for peptide fragmentation, and (b) products of hemolytic radical cleavage of an example peptide; however, the location of the added proton is not specified.
Therefore, identification is generally made by comparing measured data with theoretical data from a database of known protein sequences, and successful identifications usually rely on the availability of such databases. However, database errors, sample mutations, and the presence of post-translationally modified peptides or proteins in the sample may reduce the likelihood of identifying those peptides and proteins.
Another ion fragmentation or activation method, electron capture dissociation (ECD), is believed to involve the capture of an electron by a multiply-charged protein ion leading to the formation of a hydrogen atom that subsequently induces cleavage between the backbone nitrogen and α-carbon. See, e.g., R. A. Zubarev, N. L. Kelleher, F. W. McLafferty, J. Am. Chem. Soc. 120:3265 (1998); N. A. Kruger et al., Int. J. Mass Spectrom. 182/183:1 (1999). ECD is capable of inducing localized excitation; however, dissociation should subsequently occur before the internal energy is randomized. It has been observed that c and z type fragment ions are generated rather than the typical b and y type fragment ions. This ECD phenomenon has been used in the sequencing and identification of protein ions in specialized mass spectrometers. See, e.g., N. L. Kelleher et al., J. Am. Chem. Soc. 121:806 (1999). However, ECD cannot be used in many mass spectrometers because they contain electric fields that manipulate and control the movement of the ionized sample and sample fragments. Those fields interfere with the introduction of the electron beams that are necessary for ECD.
Bond-selective chemistry has been a subject of research by photochemists since the development and availability of tunable laser light sources. However, such bond-selective chemistry has not been applied to relatively large molecular systems or molecular systems having relatively high molecular weights, such as peptides and proteins, in part because rapid intramolecular vibrational relaxation appears to redistribute energy throughout large and/or high molecular weight molecules and their molecular ions on timescales faster than the dissociation needed for the analyses. Thus, the bond-breaking selectivity that is desired by using bond-selective chemistry in such an excitation process is often lost.
Other conventional methods for peptide ion fragmentation use an array of different activation methods, such as blackbody radiation, infra red (IR) multiphoton excitation, UV laser excitation, and collisions with gas phase molecules or surfaces. See, e.g., W. D. Price, P. D. Schnier, E. R. Williams, Anal. Chem. 68:859 (1996); J. A. Zimmerman, C. H. Watson, J. R. Eyler, Anal. Chem. 63:361 (1991); D. P. Little, J. P. Speir, M. W. Senko, P. B. O'Connor, F. W. McLafferty, Anal. Chem. 66:2809 (1994); W. D. Bowers, S. Delbert, R. L. Hunter, R. T. McIver, J. Am. Chem. Soc. 106:7288 (1984); S. A. Martin, J. A. Hill, C. Kittrell, K. Biemann, J. Am. Soc. Mass Spectrom. 1:107 (1990); D. C. Barbacci, D. H. Russell, J. Am. Soc. Mass Spectrom. 10:1038 (1990); D. F. Hunt, W. M. Bone, J. Shabanowitz, J. Rhodes, J. M. Ballard, Anal. Chem. 53:1704 (1981); E. R. Williams, K. D. Henry, F. W. McLafferty, J. Shabanowitz, D. F. Hunt, J. Am. Soc. Mass Spectrom. 1:413 (1990). These activation methods involve vibrational excitation of the precursor ion; however, the resulting peptide bond cleavages tend to produce very similar types of daughter ions, generally referred to as b and y type fragments. See, P. Roepstorff, J. Fohlman, Biomed. Mass Spectrom. 11:601 (1984).
Therefore, there is a need for methods and devices that are useful for fragmenting large and/or high molecular weight ions, including peptide and protein ions, that may lead to more predictable and interpretable results.